Hello
(I will say my genes of interest as A,B and C)
Based on the results so far, A seems to induce translocation of B into nucleus from cytoplasm. As for B, B is already known as a transcriptional regulator of C.
For me, i hypothesized that A enhances nucleus localization of B, which finally upregulates mRNA level of C and finally protein level of C.
Here is my dilemma. I am doing experiments described above under high cell confluency, but I cannot see positive regulation of C by B under such condition. (B localization depends on cell confluency, so B is mainly located in cytoplasm in dense cell condition while it is mainly found in nucleus when cells are cultured under sparse confluency)
I think that because transcription activity of B is very low in dense cell condition, it is difficult for me to confirm positive correlation between B and C expression. The positive side is that i could confirm the outcomes which support that B upregulates C
in sparse condition.
i want to ask whether i can make a research story like this
: I got results which indicate that A acts as positive upstream molecule of B, which increases C expression so A regulates C under high cell confluency.
However, because transcription activity of B is very low under such dense cell density which makes demonstrating increased C expression by B very difficult, i induced enhanced transcription activity of B by seeding cells sparsely, and i could see that B really promotes C expression. A seems to regulate C not only via B but also other factors in high cell confluecy.
For me my idea flow seems quite reasonable but i am concerned about my
quite subjective view. So I want to get some opinions from others.
Thank you for reading so long question!
This is an interesting observation!
I encourage you to spend more time on this issue to make sure your finding.
It seems that the relationship between A, B and C is context dependent. If it is true, it can serve as a very good example of the emergent properties.
Please keep me posted on this issue.
Henry Heng, Ph.D.
Professor
Center for Molecular Medicine and Genetics;
Wayne State University School of Medicine
Detroit, MI 48201 U.S.A.
Web: http://genetics.wayne.edu/heng/lab/Home.html
New Book: Debating Cancer: The paradox of cancer research
http://www.worldscientific.com/worldscibooks/10.1142/8879#t=aboutBook
Hi Lee,
It seems like you are working with some kind of epithelial cells; where cell - cell contact could reflect in the localization of certain protein inside the cell. I have a very basic points here, first is that "does your protein B is having any strong interaction with cell-cell adhesion complex? If Yes, then "does the expression of C is dependent/independent of the interaction of B to the cell-cell adhesion complex" ?.
Good luck with your findings!
Hi Lee,
Your hypothesis, rather interpretation seems quite reasonable considering that there already exist several examples of the context-dependent regulation ( e-cadherin- beta catenin and TCF pathway, for one). High-confluent and low-confluent conditions may trigger different cascades to drive the expression of C. It will be interesting to know whether the expression of C is induced (by A) to the same extent under different confluencies.
Hi agree with geetanjali. I feel that at high density cells are in position to form tight/adherent junctions which stabilizes other molecules and signaling cascade. If B activity is low under high density, I feel look at the phosphorylation and the type of phosphorylation involved under high and low density. At low density cells are apart with minimal cell connections therefore the nuclear localization of B indicates the property of B to regulate the proliferation and growth of cells. C is the binding partner for B and this interactions is possible in the absence of tight junctions. B is a co-transcription factor and is activated only when A is present. What I feel, wnt /beta-catenin, TGF/BMP-beta-catenin signaling will be useful to study and deriving insight.
Thanks
Dear Lee
The situation you describe is a general mechanism that couples cell density to transcription. This is particularly important during embryonic development, where tissues and organs develop in a coordinated fashion to reach the expected sizes and volumes. Hippo is probably the best known density-dependent payhway. At low cell density, the nuclear YAP/TAZ transcription factors activate pro-proliferative and anti-apoptotic genes. At high cell density, YAP/TAZ are phosphorylated and are retained in the cytoplasm or degraded. YAP/TAZ also interfere with growth-factor dependent pathways. A lot of articles and tools are available for Hippo.
Best wishes,
Philippe
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